Methods for Identifying Stem Cells Based on Nuclear Morphotypes

ABSTRACT

Methods for identifying stem cells and other cells specific to embryogenesis and carcinogenesis, classifying tissue samples, diagnosing precancerous and cancerous or atherosclerotic lesions, testing the value of anticancer agents, discovering macromolecules specifically expressed in particular cell types, using stem cells in restorative tissue therapy as well as methods for preparing tissue samples so heteromorphic nuclear morphotypes remain intact are disclosed.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 13/914,152, filed Jun.10, 2013 which is a continuation of Ser. No. 12/283,727, filed Sep. 15,2008, now U.S. Pat. No. 8,465,943, which is a divisional of U.S.application Ser. No. 11/156,251, filed Jun. 17, 2005, now U.S. Pat. No.7,427,502, which claims the benefit of U.S. Provisional Application No.60/580,575, filed Jun. 17, 2004. The entire teachings of the aboveapplications are incorporated herein by reference

BACKGROUND OF THE INVENTION

The processes that lead to growth and differentiation in animal embryos,fetuses, neonates and juveniles share certain characteristics with theprocesses that lead to growth and differentiation of cell typespreceding and creating tumors. Methods that recognize antigenicmolecules in tumors and fetuses have shown that there are many moleculesobservable in both fetuses and tumors that are not observed in adultorgans. In the nineteenth century it was argued that tumors might arisefrom residual embryonic cells in adults. Current views point to theexistence of self-renewing stem cells in which genetic changes occur inlineal descent from embryonic through adult stem cells that create tumorstem cells that give rise to tumors. The summarized argument is that asingle normal tissue stem cell could be the progenitor of preneoplasticlesions that in turn give rise to a founding stem cell for tumors thatin turn give rise to a founding stem cell for clonal metastases andtransplanted tumors. Based on the clear presence of histologicallydifferentiable cell types in preneoplastic lesions, tumors andmetastases, and by analogy to embryological growth and development, itcan be inferred that the originating stem cell of tissues, preneoplasticlesions, tumors and metastases must be capable of differentiation aswell as self-renewing growth. If the stem cell theory of tumor growth iscorrect, then there is a tremendous need to identify, sort, classify andmanipulate tumor stem cells. However, identification or isolation ofsuch cells has not been described in the art.

SUMMARY OF THE INVENTION

The present invention derives from the process of identification of andmethod for classifying organ-specific and/or tumor stem cells and otherspecific cell forms in a cell culture, tissue, pre-neoplastic lesion ortumor sample. The invention relates to the process of determining thenuclear morphotypes, the modes of nuclear division and the involvementof nuclei of particular morphotype in multicellular aggregates andmultinuclear syncytia among all cells in a cell culture, tissue,preneoplastic lesion or tumor sample and identification oforgan-specific or tumor stem cells on the basis of a particular nuclearmorphotype alone. Multiple forms (nuclear morphotypes) of clear andreproducible non-spherical nuclei in fetal tissue, preneoplastic lesions(adenomas of the colon) and neoplastic lesions or tumors(adenocarcinomas of the colon, carcinomas of the pancreas) were observedthat were absent in normal adult tissue such as colonic crypts or liverparenchyma. These morphotypes, disclosed herein, included nuclei of sizeand shapes previously unreported in the annals of histology or pathologyof human tissues.

The nuclear forms or morphotypes in fetal and neoplastic tissuesincluded the spherical and ovoid nuclear forms commonly seen in adulttissues but also presented a diverse set of reproducible morphotypesthat ranged in size from the 40 micron “sausage-shaped”, through shorter(˜8-20 micron) “cigar-shaped”, “bullet-shaped” and “kidney shaped” to“condensed spherical” nuclei of some 4 microns diameter. One remarkable,previously unreported, nuclear morphotype had the form of cups or bellswith an open “mouth” “designated “bell-shaped”. These bell-shaped nucleiwere observed in symmetric nuclear divisions resembling the separationof two stacked paper cups.

These “cup-from-cup” divisions were also remarkable in that they wereamitotic, e.g., did not involve condensation of all human chromosomesand separation as in mitosis. They were symmetrical nuclear divisionsinsofar as two apparently identical bell-shaped nuclei resulted from thecup-from-cup division. These bell-shaped nuclei were also observed toundergo amitotic asymmetric nuclear divisions in which a bell-shapednucleus appeared to give rise to an enclosed nucleus of one of the othernuclear morphotypes. This production of the original bell-shaped nucleusand a nucleus of a different morphotype is the first known visualizationof an asymmetrical nuclear division. The appearance of a heteromorphicnuclear morphotype distinct from the bell-shaped nucleus in asymmetricalamitotic cell divisions involving bell-shaped nuclei is disclosed hereinto distinctly define a cell with a heteromorphic nuclear morphotypedistinct from the phenotype of a cell with a bell-shaped nucleus.

Asymmetric nuclear division is widely considered as a necessarycharacteristic of stem cells in normal development. The discovery ofnuclear morphotypes common to both fetal tissue, preneoplasia andneoplasia bear on the hypothesis that tumors are a re-expression ofembryonic phenotypes, specifically stem cells forming clonal populationswith derived differentiated cellular phenotypes. The nuclear morphotypethat identifies a stem cell appears as a bell- or cup-shaped nucleus inwhich stained DNA creates a hollow structure easily differentiated fromall other nuclear morphotypes in a fetal tissue or tumor sample in whichstained DNA images show a nucleus fully encased by a DNA-containingstructure.

Further observations confirmed and extended the discovery thatadenocarcinomas and embryos partially share lineages of cells withdistinct nuclear morphotypes arising from what appears to be theidentical processes of symmetrical and asymmetrical nuclear divisions ofbell-shaped nuclei without the appearance of a mitotic apparatus. Whilethe picture of the appearance and disappearance of these nuclear formsfrom early embryo through fetal and juvenile growth to adult organ andreappearance in carcinogenesis is incomplete, the potential value ofthese findings in cancer prevention and therapy is of obvious importanceoffering benefits in diagnosis and treatment of cancers and otherdiseases such as atherosclerosis that also arise from slowly growingmonoclonal colonies. Benefits are expected from growing thesebell-shaped nuclei independently for applications such as, for example,restorative therapy for complex tissues and organs.

The value of the process described herein to specifically identify thesepreviously unrecognized cells as stem cells by their nuclear morphotype,modes of nuclear division and/or involvement in multi-nuclear structuresin normal and tumor tissues is clear. The claimed processes make itpossible to specifically recognize stem cells and other fetal andtumor-specific nuclear morphotypes, permit their isolation and study,and provide for their use in tests to discover which of many plausiblepreventative or therapeutic regimens for cancer are effective. Themethods described herein also provide means to discover specific stemcells for regeneration and transplantation therapies for human tissuesand organs (e.g., tissue restoration therapy).

In one embodiment, the invention is directed to a method forcharacterizing (e.g., classifying) a cell or tissue sample based onnuclear structures associated with stages of development or pathology,comprising: a) visualizing the nuclei of cells distributed throughoutthe tissue sample, wherein the tissue sample is prepared by a methodthat substantially preserves the integrity of structures ofsubstantially all nuclei having maximum diameters up to about 50microns; and b) determining the presence and/or absence of a class orclasses of nuclear morphotypes, wherein presence or absence of aparticular class is indicative of a stage of development or pathology.In one embodiment, the tissue sample is obtained by surgical excision.In a particular embodiment, the tissue sample is physically (e.g.,frozen) or chemically fixed (e.g., treated with one or more chemicalfixing agents selected from the group consisting of: alcohols,aldehydes, organic acids and combinations thereof such as, for example,methanol and acetic acid). In a particular embodiment, the tissue sampleis fixed prior to cellular degradation of nuclei. In another embodiment,the cells of the tissue sample are partially dissociated by tissuemaceration and spreading. In one embodiment, the cells or macromoleculesof the tissue sample are stained, thereby allowing visualization ofnuclei. In another embodiment, DNA is stained, thereby allowingvisualization of nuclei. In another embodiment, the tissue sample isfixed within 30 minutes of surgical removal.

In another embodiment, the tissue sample is obtained from amulticellular animal (e.g., a vertebrate, e.g., a mammal, e.g.,primates, rodents, canines, felines, porcines, ovines, bovines andrabbits). In a particular embodiment, the mammal is a human.

In a particular embodiment, the presence or absence of a particularclass or classes of nuclear morphotypes is indicative of a particularstage of development (e.g., embryonic, fetal (organogenesis), neonatal,juvenile and adult stages of development). In another embodiment, thepresence or absence of a particular class or classes of nuclearmorphotypes is indicative of a tissue sample selected from the groupconsisting of: normal, preneoplastic, neoplastic and metastatic. Theclass or classes of nuclear morphotypes is selected from the groupconsisting of: bell-shaped, cigar-shaped, condensed spherical,spherical, oval, sausage-shaped, kidney-shaped and bullet-shaped.

In another embodiment, the methods of the present invention furthercomprise determining the spatial or numerical distribution of one ormore classes of nuclear morphotypes within the tissue sample, whereinthe spatial or numerical distributions of the one or more classes ofnuclear morphotypes further characterizes the tissue sample. Aparticular spatial or numerical distribution is indicative of a normal,preneoplastic, neoplastic or metastatic tissue.

In another embodiment of the invention, the nuclei are contained inmultinuclear syncytia or in mononuclear cells and wherein the tissuesample is adult tissue. In one embodiment, the presence of bell-shaped,cigar-shaped or bullet-shaped nuclei are indicative of preneoplasia,neoplasia or metastasis. In another embodiment, the appearance ofmultinuclear syncytia is indicative of neoplasia or metastasis. In oneembodiment, structures indicative of amitotic symmetrical nucleardivision are indicative of neoplasia or metastasis. In anotherembodiment, the presence of bell-shaped nuclei and the absence ofmultinuclear syncytia are indicative of preneoplasia. In yet anotherembodiment, the presence of non-spherical and non-oval nuclei in bloodvessel wall tissue is indicative of an incipient atheroscleroticcondition.

In another embodiment, the invention is directed to a method foridentifying a cell of interest or multinuclear syncytium of interest ina cell culture or tissue sample, wherein the cell of interest orsyncytium of interest is identified by visualizing nuclear morphology,wherein the cell of interest comprises a heteromorphic nuclearmorphotype. In a particular embodiment, the cell or multinuclearsyncytium is isolated from the tissue sample. In another embodiment, thecell of or multinuclear syncytium is isolated by microdissection, e.g.,pressure-assisted laser microdissection. In one embodiment, the cell ofinterest or multinuclear syncytium is identified in a population ofcells in culture. In another embodiment, the cell of interest ormultinuclear syncytium is identified in a tissue sample. In anotherembodiment, the cell of interest or syncytium of interest comprises oneor more amitotic nuclear division complexes. In yet another embodiment,the cell of interest is present within a multicellular aggregate ofcells. In a particular embodiment, the cell of interest is presentwithin a cluster of multicellular aggregates.

In another embodiment, the invention is directed to a cell of interestisolated or identified by the methods disclosed herein. In oneembodiment, the invention is directed to a method for using a cell ofinterest isolated or identified by the methods disclosed herein toidentify one or more macromolecular markers specific to the cell ofinterest, wherein the marker is indicative of a particular stage ofdevelopment or pathology. In a particular embodiment, the macromolecularmarker is an antigen, cell-surface marker, nucleic acid, protein,phosphorylated protein or glycosaminoglycan.

In another embodiment, the invention is directed to a method fordiagnosing preneoplasia or neoplasia comprising identification of one ormore macromolecular markers, wherein the identification of the one ormore macromolecular markers in adult tissue is indicative ofpreneoplasia or neoplasia.

In another embodiment, the invention is directed to a method ofidentifying one or more anti-tumorigenic agents comprising: a) treatinga mammal having a tumor with one or more candidate agents; b)determining the nuclear morphology of cells contained within a tumorsample obtained from the mammal; and c) comparing the nuclear morphologyof the cells from the mammal treated with the candidate anti-tumorigenicagent with cells obtained from a mammal having a tumor but not treatedwith the candidate anti-tumorigenic agent, wherein elimination of cellscomprising neoplastic nuclear morphotypes is indicative of theeffectiveness of the agent as an anti-tumorigenic agent. In a particularembodiment, the neoplastic nuclei are selected from the group consistingof: bell-shaped nuclei, cigar-shaped nuclei and bullet-shaped nuclei. Inanother embodiment, the alteration in nuclear morphology comprises theelimination of bell-shaped nuclei. In one embodiment, the mammal is arodent (e.g., a rat or mouse. In a particular embodiment, the nuclei arearranged in syncytia and the elimination of neoplastic nuclearmorphotypes comprises a disruption of the syncytia.

In another embodiment, the invention is directed to a method ofidentifying one or more anti-tumorigenic agents comprising treating acultured tumor tissue or cell sample with one or more candidate agentsand evaluating the nuclear morphology of cells contained in the tumorsample, wherein the cells comprise heteromorphic nuclear morphotypes,wherein in the absence of an anti-tumorigenic agent, the cultured tumorcells maintain their heteromorphic nuclear morphotypes, and wherein theelimination of neoplastic nuclear morphotypes is indicative of theeffectiveness of the agent as an anti-tumorigenic agent, and wherein theelimination of preneoplastic nuclear morphotypes is indicative of atumor preventative agent. In a particular embodiment, the alterednuclear morphology comprises the elimination of one or more nuclearmorphotypes selected from the group consisting of: bell-shaped nuclei,cigar-shaped nuclei and bullet-shaped nuclei. In one embodiment, thealteration in nuclear morphology comprises the elimination ofbell-shaped nuclei. In one embodiment, the nuclei are arranged insyncytia and the elimination of neoplastic nuclear morphotypes comprisesa disruption of the syncytia.

In another embodiment, the invention is directed to a method forpreparing a mammalian tissue sample suitable for the identification ofcells comprising nuclei having maximum diameters up to about 50 microns,comprising: a) disrupting cellular adhesions of cellular sheets of thetissue sample; and b) spreading the cells with disrupted adhesions ontoa hard surface, wherein the structural integrity of the nucleus of thecells remains intact, thus rendering the sample suitable for theidentification of heteromorphic nuclear morphotype cells. In oneembodiment, the tissue sample is sectioned into layers wherein thelayers obtained exceed the thickness of a cell. In another embodiment,the tissue sample forms a layer on the microscope slide of about 0.5millimeters. In a particular embodiment, the tissue sample forms a layergreater than about 50 microns. In another embodiment, cellular adhesionsof the tissue sample are chemically disrupted (e.g., by treatment with45% acetic acid). In a particular embodiment, the fixed, chemicallydisrupted tissue samples prior to spreading are about 1 mm² in area. Ina particular embodiment, the tissue sample is a human tissue sample. Ina particular embodiment, the cellular sheets are about 1 mm² in area. Inanother embodiment, the hard surface is a microscope slide.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows nuclear morphotypes observed in interphase and earlyprophase (E.P.) cells in human embryo gut, normal colonic mucosa,adenomas and adenocarcinomas. * Bell-shaped nuclei are rarely observedin adult colon. Scale bar, 5 μm.

FIGS. 2A-2C show microscopic images of embryonic gut. FIG. 2A showsfetal gut, 5-7 weeks, at low magnification (140×) with stained nuclei(left image) and phase contrast/autofluorescent images of different gutsections (right image). Distinctly non-spherical nuclei appear to bearranged in a linear order (indicated by dashed lines) that is definedby “tube-like” structures in phase contrast images. These tube-likestructures appear throughout the gut sections observed with other cellgroupings interspersed among the tubes. FIG. 2B shows a phase contrastimage (left frame) and stained nuclei image (middle) of the identicalgut section overlaid (right) to demonstrate that the apparent linearorientation of the nuclei are in fact constrained by the tube-likestructure which is itself about 50 microns in diameter. Magnification(280×) of the nuclei (middle frame) shows that these non-sphericalnuclei appear to be in the form of cups or bells. FIG. 2C showsmagnified images (1400×) of nuclei in linear array. These arrayed nucleihave a reproducible bell shape that is apparently hollow. The “head totoe” orientation of the bells is preserved in all embryonic tubesobserved, but tubes snake backwards and forwards such that paralleltubes can have locally anti-parallel bell-shaped nuclei orientation.Scale bars, 100 μm at low and 5 μm at high magnification.

FIGS. 3A-3F show amitoses of bell-shaped nuclei embryonic gut. FIG. 3A:Symmetrical amitosis: a bell-shaped nucleus apparently emerges from abell-shaped nucleus. A variety of bell-shaped morphotypes are observedbut their amitoses show indistinguishable bell-shaped morphotypiccharacteristics such as the ratio bell mouth width/length as shown inthese three examples; FIG. 3B: Asymmetrical amitosis: a solid nuclearform apparently emerges from within the bell-shaped nucleus. Shown arefour images in which a spherical condensed nucleus is seen to be formeddeep in the bell, and apparently emerges with nucleoplasmic connectionsto the bell before separating completely; FIG. 3C: spherical uncondensednucleus coming out of the mouth of the bell-shaped nucleus; FIG. 3D:egg-shaped (“oval”) nucleus emerging from bell; FIG. 3E: bean-shaped(“kidney-shaped”) nucleus emerging from bell; FIG. 3F: cigar-shapednucleus emerging from bell. Scale bar, 5 μm.

FIGS. 4A-4C show normal adult colonic crypts. FIG. 4A shows crypts ofabout 2000 spherical and ovoid nuclei occasionally (<1/100) contained arecognizable bell-shaped nucleus (arrow in lower left corner) located atthe bottom of the crypt (right corner: enlarged image of the nucleus).FIG. 4B shows the crypt base with another bell-shaped nucleus amongother nuclear morphotypes peculiar to the base. FIG. 4C shows variousmorphotypes of interphase and mitotic nuclei of the walls and luminalsurface in a well-spread crypt. The enlarged images show: (i) sphericaland ovoid interphase nuclei, (ii, ii) early prophases of spherical andoval-shaped nuclei, and (iv) an anatelophase nucleus. Scale bars, 100 μmfor low and 5 μm for high magnification images.

FIGS. 5A-5E show enlarged images of adenomas. FIG. 5A shows a largebranching crypt characteristic of adenomas. Crypt bases and luminalopenings are regularly arranged in a manner similar to normal colonicsections. FIG. 5B shows an irregular crypt-like structure that was alsoobserved throughout the adenoma samples. Typically two, but sometimesone, four or even eight bell-shaped nuclei (insert) appear at the baseof these large (>4000 cell) irregular crypts. FIG. 5C shows a cluster ofcells of similar nuclear morphotype containing one bell-shaped nucleus.These simple clusters contain 16, 32, 64, and 128 total cells. Leftpanel: Feulgen-Giemsa stain. Right panel: phase contrast autofluorescentimage. FIG. 5D illustrates different contexts in which bell-shapednuclei appear in adenomas: (i) cluster with 31 ovoid nuclei and onebell-shaped nucleus, (ii) multiple bell-shaped nuclei in“shoulder-to-shoulder” arrangement, (iii) bell-shaped nuclei arranged in“shoulder-to-shoulder” pattern (arrowed) in larger “circles”, (iii)irregular mixture of ˜250 nuclei of with several bell-shaped nucleisuggestive of nascent crypt bases found scattered throughout adenomas.FIG. 5E shows an irregular crypt-like structure containing apparentlyclonal patches of cells of five different nuclear morphotypes with onebell-shaped nucleus (arrow) at the base. Scale bar, 100 μm for low and 5μm for high magnification images.

FIGS. 6A-6C show enlarged images of adenocarcinomas. FIG. 6A shows verylarge crypt-like structures (>8000 cells), with branches with frequentbreak points. The bases of these structures were indistinguishable fromthose of normal colonic crypts except for the presence of two(typically) bell-shaped nuclei as in adenomas. The base to lumenorientation of crypt-like structures preserved in adenomas is notobserved in adenocarcinomas in which crypt orientations appear to berandom. The arrow indicates a small ˜250 cell crypt-like structurecommonly found near the surface of the tumor. FIG. 6B shows shoulder toshoulder groupings of bell-shaped nuclei found throughout the tumorinterior. FIG. 6C shows the interior tumor mass with multiple examplesof bell-shaped nuclei locally oriented in both shoulder to shoulder andhead to toe configurations. The head to toe orientation is found only inthe tumor interior, the shoulder-to-shoulder orientation found bothinteriorly and near the tumor surface among the crypt-like structures.Bell-shaped nuclei account for ˜0.2% of all nuclei in the tumorinterior. Scale, 100 μm for low and 5 μm for high magnification images.

FIGS. 7A-7E are enlarged images depicting amitoses in adenocarcinomas.FIG. 7A shows symmetrical amitoses creating two bell-shaped nuclei ofdistinct morphologies in irregular crypts. The arrow in the left imageindicates the bottom of the bell of the newly arising nucleus. FIG. 7Bshows asymmetrical amitosis creating a spherical nucleus. FIG. 7C showsasymmetrical amitoses creating oval-shaped nucleus. FIG. 7D showssymmetrical amitoses forming “cigar”-shaped nuclei. FIG. 7E showsasymmetrical amitosis creating a “sausage”-shaped nucleus with curiousdark staining element (upper arrow) distinct from what appear to becondensed chromosomes at the lip of the bell-shaped nucleus (lowerarrow). Scale, 5 μm.

FIGS. 8A-8L shows Feulgen-DNA stained nuclei of human colonicepithelium: a, b: the bell-shaped nuclei of gut; c: of colon adenoma; d:symmetrical nuclear fission of the bell-shaped nucleus in gut; e:asymmetrical nuclear division; f: asymmetrical nuclear fission(condensed spherical nucleus emerges from the bell-shaped nucleus) incolon adenocarcinoma; g: bell-shaped nuclei at the bottom of normallooking crypts (arrowed) and h: throughout ‘incipient’ crypt of adenoma;i: diversity of nuclei morphotypes in gut. The ‘cigar’-shaped nucleiarrowed in cell spreads obtained from adenoma (k) and colonadenocarcinoma (l). In ‘j’ the nuclei of spherical shape, typical foradult normal colonic crypts, are shown. The bar size is 5 μm.

FIGS. 9A-9B shows a summary of key images. (a) Examples of nuclearmorphotypes observed in interphase and early prophase (E.P.) cells inhuman fetal gut, normal colonic mucosa, adenomas and adenocarcinomas.(H-Bell-shaped nuclei are rarely observed in adult colon). (b) Highresolution image (1400×) of bell-shaped nuclei of fetal gut. CondensedDNA appears to create an anulus that maintains an opening into thehollow bell structure. Scale bar, 5 μm.

FIGS. 10A-10B shows sections of embryonic gut, 5-7 weeks: (a) Phasecontrast image (left frame) and stained nuclei image (middle) and themerged image (right) show the linear arrays of nuclei within ˜50 microndiameter tubular syncytium; (b) High resolution image of the nucleishows hollow bell-shaped structures. The ‘head to toe’ orientation ofthe bells is preserved in all embryonic tubes observed but tubes snakebackwards and forwards such that parallel tubes may have locallyanti-parallel bell-shaped nuclei orientation. Scale bars, 50 μm at lowand 5 μm at high magnification.

FIGS. 11A-11D shows nuclear fission of bell-shaped nuclei in fetal gut.a,b: Symmetrical nuclear fission: bell-shaped nuclei emerges frombell-shaped nuclei of similar shape. c,d: Asymmetrical nuclear fission:a spherical nucleus, and a cigar-shaped nuclei emerging from abell-shaped nucleus. Scale bar, 5 μm.

FIGS. 12A-12C shows normal adult colonic crypts: (a) Crypts of about2000 spheroid, spherical or discoid nuclei occasionally (<1/100)contained a recognizable bell-shaped nucleus [arrow] located at thebottom of the crypt; (b) Crypt base showing another bell-shaped nucleus;(c) Morphotypes of interphase and mitotic nuclei of the walls andluminal surface in a well-spread crypt. The enlarged images show: [i]spherical and oval interphase nuclei, [ii, iii] early prophases ofspherical- and oval-shaped nuclei, and [iv] an ana-telophase nucleus.Scale bars, 100 μm for low and 5 μm for high magnification images.

FIGS. 13A-13E shows adenomas. (a) Characteristic large branching cryptof adenomas. (b) An irregular crypt-like structure found throughoutadenomas. Typically two, but sometimes 1, 4 or even 8, bell-shapednuclei (insert) appear at the base of these large (>4000 cell) irregularcrypt-like structures. (c) A cluster of cells of similar nuclearmorphotype containing one bell-shaped nucleus. These forms of clusterscontain exactly 16, 32, 64, and 128 total cells. Left panel,Feulgen-Giemsa stain. Right panel, phase contrast autofluorescent image.(d) Contexts in which bell-shaped nuclei appear in adenomas: (i) Clusterwith 31 ovoid nuclei and one bell-shaped nucleus, (ii) Multiplebell-shaped nuclei in shoulder to shoulder arrangement, (iii)Bell-shaped nuclei arranged in a side-by-side pattern (arrow) (iii)Irregular mixture of ˜250 nuclei of with several bell-shaped nucleisuggestive of nascent crypt bases. (e) Irregular crypt-like structurecontaining apparently clonal patches of cells of 5 different nuclearmorphotypes with one bell-shaped nucleus [arrow] at the base. Scalebars, 100 μm (in ‘a,b’) and 5 μm (in ‘e’).

FIGS. 14A-14E shows adenocarcinomas. (a) Very large crypt-likestructures (>8000 cells), with branches with frequent break points. Thearrow indicates an example of an ˜250 cell crypt-like structure foundprimarily near the surface of the tumor. (b) Interior tumor mass withmultiple where multiple bell-shaped nuclei (2 10⁻³ of all nuclearmorphotypes). (c) Bell shaped nuclei in (b) oriented in head-to-toesyncytial and non-syncytial side-by-side configurations. (d) Symmetricalnuclear fission in adenocarcinoma. (e) Asymmetrical nuclear fission of abell creating a cigar-shaped nucleus in adenocarcinoma. Similarstructures have been observed in colonic metastases to the liver. Scalebar, 5 um.

FIGS. 15A-15D shows morphological similarity of bell-shaped nuclei(Feulgen DNA stained in purple) as revealed in human tissues of: (a)embryonic gut, (b), colonic adenocarcinoma, (c) liver metastasis ofcolonic tumor, (d) pancreatic tumor. Condensed chromatin streak seen inlower half of bell in ‘d’ is seen in all bell-shaped nuclei frompancreatic but not at all in colonic tumors. Bar scale, 5 microns.

FIGS. 16A-16B shows bell-shaped nuclei detected readily by thehistological procedure disclosed herein (Feulgen stain) and rarely (onlyexample as yet) by standard histological procedures creating microtomesections of 5 microns sections (hematoxylin and eosin stain (H&E)). Sametumor, fixation within 30 minutes of resection: (a) The bell-shapednuclei giving rise to cigar-shaped nuclei (×100 magnification image tothe left) present as a colony at the edge of a pancreatic tumorcontaining a number of asymmetrically dividing cells (arrowheads). (b)The single example found to date of bell- and oval-shaped nuclei visibleon a standard tissue section slide in juxtaposition suggesting a recentasymmetrical nuclear division. The bell-shaped nucleus appears to havechromatin strands still attaching it to the oval nucleus (arrows). (c)Cartoon of the original picture showing this interpretation.

FIGS. 17A-17B is an illustration of a ‘target of interest’ inapplication of FISH to explore non-dividing and dividing bell-shapednuclei in tumors: (a) Chromatin, stained darker because of higherconcentration of DNA per square micron in the nuclear, creates theunique structure as a part of bell-shaped morphology, resemblingprophase chromosomes arranged as two parallel circles. These circles putinto drawing (above) illustrate the prediction of that specificchromosomes might be found at this specific site of bell-shaped nucleiin colon tumors, (b) Chromatin distribution and specific chromosomepositioning changes as imaginary transformation (bell-to-oval shapednuclei here) taking place throughout asymmetrical division of thebell-shaped nuclei.

FIGS. 18A-18D shows the results of fluorescent in situ hybridization ofchromosome 11 in spherical nuclei of TK-6 human cells. (a) two pairs ofchromosomes in prophase chromosome spreads, (b) spherical nuclei DAPInuclear stain, (c) same chromosome pair hybridized with FITCfluorescence probe, (d) merged image of DAPI and FITC interphasechromosomes stain.

FIGS. 19A-19C shows bell-shaped nuclei as ‘targets’ in collection bylaser pressure catapulting system (“laser pressure microdissection”):(a) The microscopic slide with cell spreads positioned in front ofpulsed UV A laser that is coupled to a microscope. (b) Single nuclei canbe seen through a microscope as shown for the cells spread of colontumor tissue with bell-shaped nuclei in vision; (c) Same nuclei withrecognizable morphology of the bell in non-stained slides (parts 1-3).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected discovery of large cellnuclei with distinct morphologies throughout fetal gut (5-7 wks),colonic adenomas, and adenocarcinomas, some of which are not present innormal (non-neoplastic) adult colon. These “heteromorphic nuclearmorphotypes” (e.g., nuclear morphotypes that differ from the normallyspheroid or ovoid nuclei of cells in adult organs), were observed inembryonic tissues and only rarely in adult tissues. One remarkablenuclear morphotype has a nucleus shaped like a hollow bell(approximately 10-15 microns in height and approximately 7-12 microns inbell mouth diameter). These bell-shaped structures appear to dividesymmetrically by an amitotic process resembling the separation of twopaper cups. Furthermore, at least seven other nuclear forms wereobserved to emerge from bell-shaped nuclei in asymmetrical amitoses.Cells containing these derivative nuclear forms subsequently divide bymitoses forming clonal populations of identical nuclear morphotypes inembryos, adenomas and adenocarcinomas. Cells with bell-shaped nucleithus appear to be responsible for both net growth and differentiation inembryonic gut, adenomas and adenocarcinomas and fulfill the requirementsfor generative, multipotent stem cells in embryogenesis andcarcinogenesis. The specific differential occurrence of bell-shapednuclei in cells demonstrating both symmetrical and asymmetrical amitosestheoretically required for net stem cell growth and differentiation inembryonic/fetal tissue sample as opposed to adult tissue, allows for oneskilled in the art of microscopic histology/pathology to classify cellsas stem cells or not-stem cells according to their nuclear morphology.

A concept shared in both embryology and oncology is that the multiplecell types that are observed in organs and tumors derived fromparticular organs arose from precursor “stem” cells capable of netgrowth by symmetrical cell divisions in which a cell division producestwo identical precursor “stem” cells and differentiation by asymmetricalcell division producing one precursor “stem” cell and one differentiatedcell. This differentiated cell may then divide an additional number oftimes to create a large number of differentiated cells that eventuallyreach a non-dividing terminal stage followed by programmed cell death.An “extinction” division occurs when a cell differentiates into two morehighly differentiated cells.

The term “stem cell” has many different levels of definition in thescientific literature. In general it is meant to comprise the set ofcells that maintain an undifferentiated or partially differentiatedphenotype but can in certain circumstances give rise to cells withdifferent phenotypes. The use of the generic term and more specificterms used herein distinguish among types of stem cells based on thescientific observations that comprise the teachings disclosed herein.

The term “embryonic stem cell” as used herein comprises the set of cellsin the early mammalian embryo that divide by ordinary mitosis and can,upon transplantation into a uterine environment, give rise to a completeplacenta and fetus. They are thus characterized as “totipotent”. Suchcells can be cultivated ex vivo, dividing by mitosis, to form very largenumbers of embryonic stem cells—each capable of giving rise to aplacenta and fetus. It is not known whether or not any embryonic stemcells persist as such in a growing mammalian fetus, neonatal, juvenileor adult animal. It is possible that certain clusters ofundifferentiated cells found throughout tissues in adult mammalsrepresent colonies of totipotent embryonic stem cells. With regard tonuclear morphotype, this form of stem cell contains spherical or nearlyspherical nuclei.

The term “fetal juvenile stem cell” as used herein comprises the set ofcells and multinuclear syncytia observed in human fetuses by the fifthweek of gestation and in human preneoplastic, neoplastic and metastaticlesions that contain bell-shaped nuclei. As used herein, “syncytia” aremulti-nuclear structures lacking cell septa. Multiple nuclei are presentbut they are not segregated into individual compartments within thesyncytia by membranes. The fetal juvenile stem cell undergoes bothsymmetrical nuclear division creating two identical bell-shaped nuclei,and asymmetrical nuclear division creating one bell-shaped nucleus andone nucleus of the several heteromorphic nuclear morphotypes observed intissue and tumor samples such as, for example, kidney-shaped,cigar-shaped, sausage-shaped, oval or spherical nuclei that subsequentlyincrease in number by mitosis. Both symmetrical and asymmetrical nucleardivisions of bell-shaped nuclei are amitotic insofar as there is nogeneral condensation of the genome as chromosomes, no formation of amitotic spindle, and no condensation or separation of chromosomes as inprophase, metaphase, anaphase and telophase as observed for more than acentury and previously believed to be the only relevant form of nucleardivision in mammalian cells (including mammalian tumors). Symmetricaldivision permits the net growth of fetal juvenile stem cells andpresumably accounts for the growth of tissues and organs throughoutfetal, neonatal and juvenile stages of life. Asymmetrical divisions offetal juvenile stem cells permits creation of cells with alternatephenotypes and presumably accounts for the differentiated cell typesthat comprise the parenchyma of developing and growing organs. Insofaras nuclear morphotype is a recognizable characteristic of a cell and/ornucleus, cells differing in nuclear morphotype can be characterized asdiffering in cellular or nuclear phenotype without reference to acharacteristic other than nuclear morphotype.

The term “adult maintenance stem cell” as used herein comprises thetheoretical form of cell that by regular asymmetrical cell divisioncreates a first transitional cell that, by subsequent mitotic divisionand ultimately programmed cell death, defines the differentiatedturnover unit of differentiated cell tissues such as, for example, thecolonic crypts. The same asymmetrical division of the adult maintenancestem cell would also create a new adult maintenance stem cell. As veryfew colonic crypts have been observed with the bell-shaped nucleicharacteristic of the observed fetal juvenile stem cells in the base ofthe crypts, it is reasonable to conclude that fetal juvenile stem cellnuclei undergo a metamorphosis marking the cessation of net growth ofjuvenile organs and the beginning of the adult stage of life. It isunknown whether the asymmetric divisions of adult maintenance stem cellsare mitotic or amitotic in nature.

The term “preneoplastic stem cell” as used herein describe mononuclearcells comprising the observed bell-shaped nuclei found in the base ofadenomatous colonic crypts. These cells are observed in small aggregatesof cells of identical nuclear morphotypes, in large clusters of suchaggregates in which each aggregate creating the cluster contains nucleiof the same morphotype with different aggregates contain nuclei ofdiffering morphotype, and in subregions of the colonic preneoplasticlesions. These cells can have nuclei arranged in a “shoulder-toshoulder” relationship. Syncytia containing bell-shaped nuclei have notbeen observed in human colonic preneoplastic lesions nor have theindividual bell-shaped nuclei been observed in symmetrical nucleardivision, a condition interpreted as consistent with the slow growth ofpreneoplastic lesions at approximately the growth rate of the juveniletissues from which they were derived.

The term “neoplastic stem cell” or “tumor stem cell” as used hereincomprises the bell-shaped nuclei found in cancerous tumors, e.g.,adenocarcinomas of the human colon, metastases of colonic tumors andtumors of the human pancreas. These neoplastic stem cells aredifferentiated from preneoplastic stem cells by their presence in all ofthe observed multicellular aggregates of preneoplasia, but, in addition,being found and found to be dividing by amitotic symmetrical nucleardivisions in multinuclear tubular syncytia. The syncytia found in tumorsare less extensive than those observed in early fetal gut tissue, butthe syncytia and bell-shaped structures undergoing symmetrical nucleardivisions appear to be clear distinguishing characteristics betweenpreneoplastic and neoplastic tissue.

The heteromorphic nuclear morphotypes described herein can also be foundin cells and syncytia derived from, for example, a tissue sample orcells grown in culture. The methods described herein can be applied toany tissue sample to identify cells or syncytia containing theheteromorphic nuclear morphotypes. In particular, tumor tissue samplescan be used to identify and enumerate heteromorphic nuclear morphotypes.

As used herein, “tumor” refers to an abnormal growth of tissue resultingfrom progressive multiplication of cells serving no physiologicalfunction that is beneficial to the carrier (also referred to as a“neoplasm”). A ‘benign’ tumor is a tumor limited to the site of originwithout invasion of the surrounding tissue. Malignant tumors are thosethat can or do spread by invasion of surrounding tissue and metastasiswhile benign tumors neither invade nor metastasize. As used herein,“neoplastic” refers to a cellular condition of rapid net cell growthgiving rise to a lethal tumor, whether benign or malignant. Neoplasticcells lead to tumors, although not necessarily invasive or metastatictumors. “Cancer” refers to the disease of one or more rapidly growingcolonies of cells that cause death by interfering with bodily functions.Cancer cells can break away from a primary tumor, penetrate intolymphatic and blood vessels, circulate through the bloodstream, and growin a new site (metastasize) in normal tissues elsewhere in the body.

As used herein, a “pre-cancerous condition” is characterized as a slowlygrowing colony that, if unchecked, has the potential to give rise tocancer (e.g., neoplastic tumors). This condition is characterized, forexample, by the presence of an abnormal microanatomical structure orstructures such as, for example, polyps in the colon or bell-shapednuclei within an adult tissue sample.

The “net stem cell growth rate” is the rate (divisions per unit time) ofsymmetrical fetal juvenile stem cell divisions that account for netgrowth of a tissue, organ or organism. In general stem cell net growthrates decrease with increased development of an organism throughoutfetal, neonatal and juvenile life to zero in adults.

Tumors display many of the characteristics of the adult organ/systemfrom which they are derived including formation of complexmulti-cellular structures, such as, for example, colonic crypts inadenocarcinomas of the colon. Insofar as tumors have been derived from asingle precursor cell that had the ability of both net growth anddifferentiation, it has been reasoned that “tumor stem cells” must existand share many characteristics organ-specific stem cells (partiallyundifferentiated cells capable of giving rise to specific organ cellsand tissues). For example, in the case of the cells of the bone marrowfrom which leukemias arise, certain antigens have been recognized thatallow identification of marrow cell sub-populations that contain one ora small number cells capable of giving rise to a complete blood cellsystem on transplantation. Similarly a subfraction of tumor cells can berecognized by specific antigens expressed by some tumor cells. Accordingto this logic, one cell among the sub-population so recognized givesrise to a new differentiated tumor upon experimental transplantation ofmixtures of tumor cells. Continuing with this reasoning, the“organ-specific stem cells” and “tumor stem cells” exist among suchantigen expressing sub-populations. These antigens are used to identifyand isolate cell populations containing at least one pluripotent stemcell, but will also identify a large majority of cells that cannot actas stem cells. None of the cells of these populations as isolated havebeen found to undergo asymmetrical cellular divisions that areconsidered a “shibboleth” of true stem cells or organs and tumors.

Presented here are methods for identifying cells that are in fact“organ-specific stem cells” or “tumor stem cells” as opposed tosubfractions of organs or tumors enriched for such cells. Theidentification is based on the heteromorphic nuclear morphotypes andnuclear divisions and/or arrangements described herein. With thesemethods, it would be possible to isolate such stem cells and discovertheir specific biochemistry and molecular biologies with the goals ofisolating cells permitting organ regeneration, interfering with thegrowth of pre-cancerous lesions and/or the killing of the cellsspecifically responsible for tumor growth and reappearance afterattempts at therapy.

Such organ-specific and tumor stem cells are further identified in fetalorgans, tumors and tumor metastases by their participation as a class intwo specific previously unreported, microscopically visible forms ofnuclear division. In the first of these two forms of nuclear divisionthere is no general condensation of chromosomes and formation of amitotic apparatus; instead one bell-shaped nucleus appears to give riseto an identical bell-shaped nucleus in a manner similar to separation oftwo paper cups. This symmetrical, amitotic division provides for the netgrowth of stem cells in fetal and tumor samples. In the second form ofstem cell-specific nuclear division there is also no generalcondensation of chromosomes and formation of a mitotic apparatus;instead one bell-shaped nucleus appears to give rise to a bell-shapednucleus and a nucleus of a nucleus having one of the several nuclearmorphotypes observed in fetal tissue and tumor samplers (FIG. 3). Thisasymmetrical, amitotic division provides for creation of differentiatedcells by stem cells in fetal tissue and tumors. Mitotic division ofnuclei without bell-shaped nuclei are observed in fetal tissue and tumorsamples creating the majority of total cells in such samples. Thus theprocess of identification of stem cells permits direct observation ofcells with bell-shaped nuclei undergoing both symmetrical andasymmetrical nuclear divisions, both necessary for classification asorgan-specific and tumor-specific stem cells.

Such organ-specific and tumor stem cells are further identified in fetalorgans, tumors and tumor metastases by their participation as a class inspecific previously unreported multi-nuclear structures resembling longtubes in which bell-shaped nuclei are regularly aligned in a fashionresembling a series of separated paper cups retaining the head-to-toerelationship as in the stacked cup set.

These stem cell-specific nuclear morphotypes (bell-shaped), specificforms of nuclear division (symmetrical and asymmetrical amitotic nucleardivisions) and specific form of participation in multinuclear structures(long multinuclear tubes) are essentially absent from adult organs.However, the stem cell-specific nuclear morphotype is observed among thecells of preneoplastic lesions and rarely, as single nuclei, widelydispersed in adult organs (for example, somewhat fewer than one in twomillion nuclei of adult colonic crypts have been found to be bell-shapednuclei).

The unexpected discovery of these heteromorphic nuclear morphotypes andtheir differential occurrence in stem cells, normal adult tissue andtumor tissue allows for one skilled in the art to classify cellsaccording to their nuclear morphology. In addition, stem cells can beisolated based on their nuclear morphology, anti-tumorigenic agents canbe screened or identified based on the appearance or disappearance oftumor-specific morphotypes, and tissue samples can be classified (e.g.,normal or abnormal; neoplastic or non-neoplastic) by determining themorphotypes present in the cells of the tissue. As a result of this cellclassification, a diagnosis can be made as to whether or not anindividual has cancer or a pre-cancerous lesion. Although a particularmethod (see below) is used in the Examples to allow for thevisualization of these novel morphotypes, any method that allows for theidentification and evaluation of nuclear morphotypes is suitable for theclassification of cells, tissues and samples, and subsequent diagnosisof disease, as well as provide the basis for assays used to identifyanti-tumorigenic agents (e.g., agents effective in inhibiting ordecreasing tumorigenic cell growth)—. Such methods include phasecontrast microscopy, confocal microscopy, two electron or two wavelengthmicroscopy and small angle scatter flow cytometry.

For the purposes of the present invention, thick, ˜0.5 mm, sections ofnormal adult human colonic epithelium, colonic adenomas, colonicadenocarcinomas, and fetal gut were prepared for microscopic observationas described in the Exemplification below. The thickness of the tissuesheet layer should be at least the thickness of an intact cell (and notjust a section or slice of the cell). An array of large spheroidal andnon-spheroidal nuclear forms appeared in all samples as summarized inFIG. 1. All of the samples contained the spheroid and ovoid nucleinormally observed in histological sections of adult colonic crypts butalso contained extraordinary, previously unreported nuclear morphotypes.Embryonic tissue contained nuclei shaped like bells, tapered cigars,kidney beans, sausages and small spheres. Normal adult colonic cryptscontained an occasional bell-shaped nucleus in the crypt bases but thevast majority were the large spheres and ovoid structures. In some viewsit appears that cell nuclei near the crypt bases may be “discoid”. Inadenomas and adenocarcinomas the nuclear shapes, in addition to thespheroid and ovoid nuclei, included tapered cigars and an additionalform that looks like a cigar with a bitten off end dubbed“bullet-shaped”.

Crypt structures were preserved by the preparative procedure and wereclearly observed in normal colon, adenomas and adenocarcinomas. The ˜0.5mm sample sections employed were much thicker than the largest nuclearform observed, the sausage-shape, which was ˜40 microns in length. Allof the nuclear structures, except the 4 micron “condensed sphericalnuclei”, had at least one internal axis longer than the 5 micronsections usually employed in pathological evaluations. Furthermore,there is a fair degree of morphological variation among nuclei that canbe classified as “bell-shaped” or “cigar-shaped” etc., suggestingindependent lineages and physiological functionalities.

The phenomena of bell-shaped nuclei, their symmetric and asymmetricforms of amitosis, or the collection of nuclear morphotypes in adult,preneoplastic, neoplastic and embryonic tissue described herein have notbeen previously reported (see Example 2). The tubular encasement oflinearly arrayed bell-shaped nuclei in embryos and adenocarcinomas isalso apparently a novel observation. The reason that they have not beenpreviously observed may lie in the differences between standardhistological practices and those employed and disclosed herein. Twoclear procedural differences are evident. First, all tissues forfixation were sectioned and fixed within a short period of time (forexample, within 30 minutes) of surgical removal. Preparations after 30minutes may begin to show degradation of the nuclear forms, althoughcareful tissue preparation may prevent degradation. Second is thedifference between thin section procedures practiced in medicalpathology and thick section fixation protocols, disclosed herein—thelatter preserving, and the former apparently destroying, thestructures/conditions that maintain these nuclear shapes.

Amitosis, as a phenomenon, has been reported in a number of protozoansand primitive metazoans (Orias, E., 1991, J. Protozool., 38:217-221;Prescott, D., 1994, Proc. Natl. Acad. Sci USA, 92:136-140). However,these amitoses were unlike those reported here insofar as protozoanamitotic nuclear division occurred by formation of a nuclear cleft andpinching off two separate approximately equal nuclei (Fujiu, K. andNumata, O., 2000, Cell Motil. Cytoskeleton, 46:17-27). Amitoticdivisions of the sort similar to those seen in protozoans have beenreported, however, in a number of different tumors (Okuyama, S. 1991,Tohoku J. Exp. Med., 164:247-249; Okuyama, S., 1992, Tohoku J. Exp.Med., 168:445-448; Elias, H. and Fong, B., 1978, Hum. Pathol.,9:679-684; Elias, H. and Hyde, D., 1982, Hum. Pathol., 3:635-639).

The observations showing that the arrangement of chromosomes in earlyprophase nuclei of the mitotic cells maintains the shape of theinterphase nucleus also deserve attention. It appears that the differentchromosomes form a highly structured mosaic that may have importantconsequences in defining a cell's phenotype. The relationship betweenthe spatial arrangement of chromosomes in interphase nuclei and cellphysiology is an active area of exploration (Misteli, T., 2001, Science,291:843-847; Thomas, C. et al., 2001, Proc. Natl. Acad. Sci. USA,99:1972-1977; Parada, L. et al., 2004, Exp. Cell Res., 296:64-70).

Based on the observations cited below, cells with bell-shaped nuclei arepluripotent cells that represent the generative cell of the developingand growing tumors and preneoplastic lesions such as, for example,colorectal tumors, adenomas and adenocarcinomas. Thus, the bell-shapednuclear morphotype is indicative of pluripotent stem cells and can beused as diagnostic criteria for preneoplastic and neoplastic tissue inadult tissue samples. In addition, the organization of bell-shapednuclei (e.g., into tube-like structures or spider-web-like structures)can be further indicative of the progression of tumor development (e.g.,neoplasia or metastatic tumors).

The pluripotency of bell-shaped nuclei is demonstrated by the images ofmultiple forms of nuclei emerging from bell-shaped nuclei inasymmetrical amitotic divisions. Insofar as egg-, sausage, kidney-,bullet- and cigar-shaped nuclei are observed emerging from bell-shapednuclei and no other nuclear forms are observed, they represent the setof functions necessary for the tissue in which they reside to persist.There can indeed be multiple forms of cells with bell-shaped nuclei assuggested by the morphological variations among bell-shaped nucleiobserved.

The numbers and symmetrical amitotic frequencies of cells withbell-shaped nuclei are consistent with the generative element of thishypothesis. They are observed in large numbers in embryos, rare innormal adult colon free of neoplasia, present, in small numbers (forexample, about 1,000) in adenomas of a few cubic millimeters and largenumbers (for example, less than about 1,000,000) in adenocarcinomas ofseveral cubic centimeters. Their division rates in the embryo andadenocarcinomas are estimated to be approximately 20 divisions per yearconsistent with the estimated net growth rates of colonicadenocarcinomas (Herrero-Jimenez, P. et al., 1998, Mutat. Res.,400:553-578). Their symmetrical amitotic fraction in adenomas is lessthan 1/1000 and none have been seen to date. This negative observationis important in itself. The frequency of cell divisions for thegenerative cell or “cell at risk of promotion” in human colonicpreneoplastic lesions has been estimated by calculation to be about onein six years (Herrero-Jimenez, P. et al., 2000, Mutat. Res.,447:73-116). Assuming amitosis could be recognized for a three hourperiod, a frequency of less than 6/100,000 would be expected. Thus, thevery low symmetrical amitotic rate of adenomas is consistent withexpectation for the generative cells of colonic preneoplasia.

It is possible that the cells with bell-shaped nuclei are phased out atthe end of juvenile growth. Retinoblasts phase into retinocytes in earlychildhood and remove the risk of retinoblastoma in retinoblastoma geneheterozygotes (Knudson, A., 1971, Proc. Natl. Acad. Sci. USA,68:820-823). It could be that colon tumor “initiation” by mutations ingenes such as APC prevents this phasing out process. If so, bell-shapednuclei should be found in the bases of colonic crypts in neonates andjuveniles.

From the appearance of bell-shaped nuclei in embryonic and carcinogenictissues, relationships between embryogenesis and carcinogenesis can beinferred. Cancer researchers have considered tumors to reflectcharacteristics of embryos for more than a century. Erenpresia, J. andHelmtrud, I. (1999, Mech. Aging and Develop., 108:227-238) cited J.Cohnheim (1875, Virchows Arch., 65:64; 1877-1880, Vorelesungen uberallgemeine Pathologie. Ein Handbuch fur Artzte and Studierende. Berlin,Hirchswald 1-2 691 S) as first hypothesizing that tumors arise fromfetal cells that inappropriately persist in adult tissues. Theexpression of carcino-embryonic antigens in tumors and appearance intumors of a wide spectrum of gene products, mRNAs and proteins, that arealso found in embryos has reinforced the broad hypothesis thatoncogenesis involves the appearance of cells with embryo-like qualities.The finding of morphological cell types essentially identical in form,amitotic and mitotic behavior in adenocarcinomas and embryonic coloncalls for a more specific restatement of the carcino-embryonichypothesis in terms closely echoing Cohnheim and using the more recentarguments and experimental demonstrations indicating the existence oftumor stem cells (Pardal et al., 2003, Nature Rev., 3:895-902).

These new observations, integrated into the body of cancer research andideas of the past 130 years, suggest a simple hypothesis about theorigin and characteristics of late-onset colonic adenomas andadenocarcinomas: tumor initiating mutations, e.g., APC geneinactivations, occur in a cell with a bell-shaped nucleus before thiscell form is phased out during or at the end of the juvenile period.Such initiated cells with bell-shaped nuclei would simply continue todivide and create new colonic crypts at the same rate as they did injuveniles (Herrero-Jimenez, P. et al., 1998, Mutat. Res., 400:553-578;Herrero-Jimenez, P. et al., 2000, Mutat. Res., 447:73-116). Theresultant local crowding creates the “polyp”. Either actively, by anadditional genetic change or changes (including changes in geneimprinting), or passively by biochemical changes occurring within thegrowing adenoma, a single cell with a bell-shaped nucleus reverts to anearlier embryonic condition and gives rise, as in the embryo, to arapidly growing array of cells almost indistinguishable from embryonictissue. Untreated, this continued growth leads to colonic obstructionand/or metastases and death.

In the most general sense these observations point to a highly orderednature of carcinogenesis in which distinctly non-chaotic behavior isobserved in adenomas that preserve the slow but constant growth rate ofjuveniles and in adenocarcinomas that recreate an ordered ensemble ofcell types and growth rates observed during embryogenesis. In the sensethat the existing biological forms have been selected from a myriad ofdegenerate possibilities (“trying all combinations”), it is perhaps notsurprising that carcinogenesis in humans might represent a rare butsimple failure to cease juvenile growth and a subsequent rare reversionto an ordered embryonic cell state.

These observations suggest that cells with bell-shaped nuclei would betargets for more specific and therefore more effective forms of tumorprevention and therapy. Were it possible to drop the net growth rate ofpreneoplastic colonies by 50%, most late onset cancer types would notappear during a human lifetime of 100 years (Herrero-Jimenez, P. et al.,1998, Mutat. Res., 400:553-578; Herrero-Jimenez, P. et al., 2000, Mutat.Res., 447:73-116). It is reasonable to believe that cells withheteromorphic nuclear morphotypes such as, for example, bell-shapednuclei are the tumor stem cells in adenomas and adenocarcinomas of thecolon, one might target the mechanisms that confer their specialcharacteristics in DNA synthesis and segregation either in symmetricaldivisions of net growth or the asymmetric divisions that provide thecells that divide by mitosis and provide the bulk of the tumor mass. Itmay be that these cell types or entubated bell-shaped nuclei operateunder different biochemical rules and that these, if understood, mightalso be exploited in tumor prevention and/or therapy (see, for example,the findings of Otto Warburg who discovered marked differences inmitochondrial biochemistry among embryonic, adult organ and cancertissues (Warburg, O., 1956, Science, 123:309-314; Warburg, O. et al.,1960, Z. Naturforsch B., 15B:378-379).

Reference to basic texts on invertebrates shows that a largesausage-shaped nucleus exists among the ciliated protozoans such as thepertirich Vorticella and the heterotrich “Stentor has a remarkable typeof large nucleus resembling a string of beads . . . ” (Buchsbaum, R. etal., 1971, Animals Without Backbones, 3rd ed., University of ChicagoPress, Chicago, Ill.). These peculiarities of nuclear metamorphosesmight even be linked in evolutionary time with the biochemistry of cellssurviving in the pre-oxic environment insofar as they appear to grow inembryos, adenomas and adenomas in local milieu that would be expected tobe oxygen poor prior to neo-vascularization. Warburg's discovery thatamino acids provide the oxygen reducing equivalents for ATP generationin embryos and tumors suggests selection of a phenotype that treatsoxygen as the limiting nutrient.

The heteromorphic nuclear morphotypes (e.g., bell-shaped nuclei) thusappear to represent a three-fold physiological nexus unitingevolutionary biology, embryogenesis and oncogenesis. To the geneticcycle of meiosis and mitosis, symmetrical and asymmetrical amitoticstages of lineal descent between the mitotic divisions of thepost-fertilization period and the mitotic divisions that create most ofthe cellular mass of an animal must now be added.

The present invention is specifically directed to methods of classifyingcell types based on nuclear morphology, their involvement in symmetricaland asymmetrical amitoses and their association in multicellularaggregates. For example, a tissue sample obtained from a mammal can beclassified based on the presence or absence of heteromorphic nuclearmorphotypes (e.g., bell-shaped nuclei, cigar-shaped nuclei andbullet-shaped nuclei). As FIG. 1 demonstrates, heteromorphic nuclearmorphotypes are present in different stages of development and also atdifferent stages of tumor development. For example, bell-shaped nucleiare found in fetal samples, rarely in adult samples, and prevalently inadenoma and adenocarcinoma samples. This supports the notion thatheteromorphic nuclear morphotypes can be used to identify fetal juvenilestem cells as well as neoplastic stem cells in adult tissues. Theseresults demonstrate a previously undocumented link between fetal stemcells and cancer stem cells. Thus, heteromorphic nuclear morphotypesthat are indicative of fetal juvenile stem cells in fetuses are alsoindicative of cancer stem cells in adult tissues.

In addition, FIGS. 2A-2C show that heteromorphic nuclear morphotypesalign in tube-like structures. As these tube-like structures are presentin fetal samples and contain heteromorphic nuclear morphotypes (e.g.,bell-shaped nuclei, cigar-shaped nuclei and bullet-shaped nuclei), thetube-like structures themselves can be used as indicia of fetal juvenilestem cells or neoplastic (e.g., tumor or cancer) stem cells in adulttissues.

Alternatively, the arraying of heteromorphic nuclear morphotypes intosyncytia as shown in FIG. 5A-5E is indicative of differences betweenadenomas and adenocarcinomas. Therefore, the arrangement of nuclearmorphotypes in multinuclear structures in a tissue sample can be used todifferentiate among neoplastic samples at different stages of disease.

As heteromorphic nuclear morphotypes have been observed in other adulttissues and tumor samples (e.g., liver), one of skill in the art wouldrecognize a preneoplastic or neoplastic lesions based on the presence ofheteromorphic nuclear morphotypes in any adult tissue.

The present invention is also directed to methods for identifyinganti-tumorigenic agents based on the appearance or disappearance ofheteromorphic nuclear morphotypes specifically associated withpreneoplastic or neoplastic tissues (e.g., bell-shaped nuclei,cigar-shaped nuclei and bullet-shaped nuclei). Candidateanti-tumorigenic agents can be screened, for example, in vivo inparticular animal models (e.g., mammalian models, e.g., rodents such as,for example, mice or rats). Candidate anti-tumorigenic agents can bescreened, for example, in clinical studies to discover if a trialregimen actually destroys the tumor stem cell component as opposed tothe non-stem cell population that constitutes >99% of the cells in atumor. With the process described herein candidate anti-tumor agents ortumor prevention agents can be screened in experimental animals usingtransplants from human tumors or tumors that arise de novo inexperimental animals. Thus, agents that kill or interfere with thesymmetrical or asymmetrical divisions of cells with bell-shaped nucleiin cell culture would be recognized as candidates for tumor preventionor therapy in patients.

Alternatively, anti-tumorigenic agents can be screened in vitro or exvivo (e.g., in cultured samples where nuclear morphologies aremaintained). Methods for preserving tissues in primary cultures forextended periods of time are known in the art. Anti-tumorigenic agentscan be identified in cultured samples where heteromorphic nuclearmorphotypes are preserved. Thus, if such a cultured sample is treatedwith a candidate anti-tumorigenic agent and the prevalence ofheteromorphic nuclear morphotypes is diminished, then the candidateanti-tumorigenic agent would be expected to be useful in treating tumorsin patients in vivo.

The invention will be further described with reference to the followingnon-limiting examples. The teachings of all the patents, patentapplications and all other publications and websites cited herein areincorporated by reference in their entirety.

EXEMPLIFICATION Example 1 Experimental Procedures Sources of Cells andTissues

All adult tissue and tumor specimens were obtained as surgical discardsat the Massachusetts General Hospital through the Department ofPathology and the MGH Center for Cancer Research. Each tissue sectionwas immediately placed in fresh ice-cold fixative and transported to theMIT cytogenetics laboratory for further analyses. Use of the anonymousdiscarded sections had been approved by the Institutional Review Boardsof both MGH and MIT. The fetal gut sections analyzed were not obtainedfor this research but were drawn from the archival slide collection ofthe Chernobyl Scientific Expedition charged with the task of discoveringsigns of genetic radiation damage in developing fetuses and childrenafter the meltdown of the nuclear reactor at Chernobyl, Ukraine in 1985.Two normal adult colons that were discarded after surgery not related tocancer (five ˜2 to 10 mm diameter polyps of two FAPC colons, four colontumors, one pancreatic tumor and several independent colorectalmetastases of the liver from two patients) were analyzed.

Tissue Excision, Fixation, Spreading and DNA Staining

The procedure uses fixed and stained tissue sections some 0.5 mm inthickness in which cellular adhesions are chemically disrupted to adegree that permits an orderly spreading of tissue on a microscopeslide. This technique allows for the cells to retain the structuralintegrity of their nuclei. Small morphological structures such asstained nuclei and larger structures such as colonic crypts may still beobserved albeit with some minimal distortion inherent in tissuespreading.

Nuclear morphotypes are visible especially where the colonic surgicaldiscards are provided soon after resection (e.g., less than an hourafter resection, more preferably, about or less than 30 minutes afterresection). Sheets (˜1 cm²) of stripped colonic mucosa or 1 mm thicksections of adenomas or adenocarcinomas were placed immediately upondissection into freshly prepared 4° C. Carnoy's fixative (3:1,methanol:glacial acetic acid). The volume of fixative is at least threetimes the volume of the tissue sample. Fresh fixative is replaced threetimes every 45 minutes for a total of three hours of fixation. Carnoy'sfixative is then replaced by 4° C. 70% methanol and may be stored up toa year at −40° C.

About 1 mm² pieces (length×width as distinct from section thickness) areexcised from the whole fixed tissue sample for spreading and DNAstaining Each piece is rinsed in distilled water and placed in 2 mL of 1N HCl at 60° C. for precisely 8 minutes for partial hydrolysis ofmacromolecules and DNA depurination. The hydrolysis is terminated by arapid rinse in cold distilled water. The rinsed sample is steeped in 45%acetic acid (room temperature) for 15 to 30 minutes. This last step isknown in botanical cytogenetics as “tissue maceration” that allowssubsequent tissue cell spreading and observation of plant tissuesections with gentle pressure on microscope slides (Gostev, A. andAsker, S., 1978, Hereditas, 101:98-104; Gostjeva, E., 1998, Genetika,32:17-21). This “macerated” fixed tissue sample is used immediately forspreading on microscope slides. Each ˜1 mm² macerated section isbisected to form two ˜0.5×1 mm pieces of fixed, macerated tissue. Eachpiece is transferred into ˜5 μL of acetic acid on a clean microscopeslide and covered with a 22×22 mm cover slip. Holding the cover slip bythe edges slight pressure is applied on the tissue sample to locate itin the middle of the slide.

For the spreading step, 5 layers of filter paper are folded and placedon the cover slip taking extreme care not to move the cover slip. Atweezer handle is moved steadily in one direction along the filter papercovering the cover slip with slight and even pressure. “Slight” meansmarkedly less pressure than used in chromosome “squashes”. The qualityof the spreading is checked using a 20× phase-contrast objective foreach individual sample. An indication of a good colonic tissue spread isthat there are no damaged nuclei on the edges of the whole tissue spreadwhile crypts are pressed into what is essentially a monolayer preservingthe morphological integrity of the crypts. Each well-spread sample slideis placed immediately on a dry ice surface. In 2 minutes, when thespread tissue sample is completely frozen, a razor blade is insertedunder one edge the cover slip that is gently lifted off. Slides areallowed to dry in a dust free environment for not less then one hour.

Staining procedures are performed at room temperature. Slides are placedin Coplin jars and filled with Schiffs reagent (Art. 9033, Merck) tostain the partially depurinated DNA of the nuclei. Slides are immersedin staining solution for one hour, rinsed in the same Coplin jar twotimes in 2 SSC (trisodium citrate 8.8 g/L, sodium chloride 17.5 g/L),once for 30 sec and once quickly. Slides are then rinsed with distilledwater. The slides at this stage are suitable for observation of thedistribution of DNA in nuclei including measurement of Feulgen DNAamounts in nuclei or condensed chromosomes by quantitative imageanalysis (Hardie, D. et al., 2002, J. Histochem. Cytochem., 50:735-749).

To achieve superior resolution and imaging of interphase nuclei slidesmay be further stained with Giemsa. Immediately after rinsing in 2 SSCslides are placed in 1% Giemsa solution (Giemsa, Art. 9204, Merck) for 5minutes then rinsed quickly first in Sörenssen buffer (disodium hydrogenphosphate dihydrate 11.87 g/L, potassium dihydrogen phosphate 9.07 g/L)and then distilled water. Water drops are shaken off the slide as if onewere shaking a thermometer to avoid erosion of the stain. The slides areplaced in a dust free environment to dry at room temperature for onehour. They are then placed in a Coplin jar filled with Xylene for atleast 3 hours to remove fat. Cover slips are glued to the slides withDePex mounting media and permitted to dry for 3 hours at which time theyare ready for high resolution scanning.

Microscope and Image Processing System

A KS-400 Image Analysis System™, Version 3.0, (Zeiss, Germany) was usedto observe and record images for future quantitative analyses of nucleardimensions and DNA content. The system consists of a motorized lightmicroscope, Axioscope™, color CCD camera, AxioCam™ (Zeiss, Germany)linked to a personal computer. Images were transmitted from themicroscope at 1.4/100 magnification of the planar apochromatic objectiveusing visible light and 560 nm (green) filter when Feulgen stain alonewas employed. No filter was used when Feulgen-Giemsa staining wasemployed. The frame grabber and optimal light exposure were adjustedprior to each scanning session. Nuclear images were recorded at a pixelsize 0.0223 0.0223 um. Scanning parameters such as magnification,resolution and light exposure were saved to permit reproducible scans ofthe same slide.

Example 2 Detection of Bell-Shaped Nuclei Embryonic Hindgut

Observations described herein are from two independent embryo gutsections are shown in FIGS. 1, 2A-2C and 3A-3F. Three observations weremade. First, there was the array of seven distinct nuclear morphotypessummarized in FIG. 1. Secondly there was the orderly linear head to toearrangement of bell-shaped nuclei organized in long (˜20 to 50 microndiameter) tubes as shown in FIGS. 2A-2C. Thirdly, there were theextraordinary forms of symmetrical and asymmetrical amitoses involvingbell-shaped nuclei as shown in FIGS. 3A-3F.

Phase contrast images (left frame, FIG. 2B) and stained nuclear images(middle frame, FIG. 2B) of the identical hindgut section when overlaid(right frame, FIG. 2C) showed that the a linearly oriented nuclei werecontained in a previously unreported tube-like structure which is itselfabout 20-50 microns in diameter. 280× magnification of the nuclei(middle frame, FIG. 2B) shows that these non-spherical nuclei appear tobe in the form of cups or bells. Higher resolution images (1400×) ofnuclei in linear array shows them to have a reproducible bell shape thatis apparently hollow. The “head to toe” orientation of the bells waspreserved in all embryonic tubes observed but tubes snake backwards andforwards such that parallel tubes may have locally anti-parallelorientation of bell-shaped nuclei.

Bell-shaped nuclei were observed undergoing symmetrical or asymmetricalamitoses only within the tube-like structures. Symmetrical amitoses ofbell-shaped nuclei resembled a simple separation of two stacked papercups. At the highest resolution, the upper lip of thesebells-in-division appeared to have a pair of condensed or partiallycondensed chromatids (see FIG. 1, arrows) encircling perhaps ¾ths of thebell's outer rim, as seen in FIGS. 3A and 3B. A variety of differentbell shapes were found within the various tubes and these morphologicalvariations were faithfully reproduced in symmetrical amitoses, FIG. 3A.

This previously undiscovered form of amitotic nuclear division ofbell-shaped nuclei was, of course, surprising. But the fetal sectionshad more surprises than this. Throughout the gut sections bell-shapednuclei within tubular structures were found apparently “giving birth” tonuclei. Examples of every form of nuclear shape found in the fetalsections and shown in FIG. 1 were found emerging from bell-shapednuclei. These extraordinary amitotic asymmetrical forms of nucleardivision are shown in FIG. 3C.

With regard to the nuclear morphotypes other than bell-shaped, allappeared to undergo mitoses forming local colonies of identical nuclearmorphotypes. These mitotic divisions always occurred outside of the longtubes containing bell-shaped nuclei. Curiously, the specific nuclearmorphology was preserved in prophase, and even recreated by associationof the chromosomes in late anatelophases, as is also shown in FIG. 1.

Normal Colonic Epithelium

The combination of ˜0.5 mm sections, the tissue maceration (see below)and gentle spreading combined with the DNA-specific staining to createparticularly clear images of nuclei permitted recognition of threedimensional features. All, or nearly all, nuclei in crypts could beobserved from the crypt base to the luminal surface. Many crypts eitherfractured or spread in such a way that individual nuclear shapes couldbe discerned. Cells with ovoid or spheroid nuclei line the crypt fromjust above the base to the epithelial extension into the lumen. However,in the first ˜25 cells of the crypt base itself a nuclear morphotypethat may be characterized as “discoid” (˜2-3 microns thick and ˜10microns diameter) predominated. In less than 1% of crypt bases in whichthe cells were well separated a bell-shaped nucleus was discerned amongthe discoid nuclei. A similar low frequency of bell-shaped nuclei hasbeen observed in preparations of adult liver. In an adult colon withoutany indication of neoplasia or preneoplasia no other nuclearmorphological variant was observed in a cell by cell scan of about 800well spread crypts.

Adenomas

The adenomas contained many crypts, indistinguishable from normalcolonic crypts, each with ˜2000 cells. These were frequently found inbranching forms as shown in FIG. 5A. The same spheroid and ovoid nucleiwere present in the crypt walls, as in the normal colonic crypts, butfrequently displayed one or two bell-shaped nuclei in the crypt base.Irregular crypt-like structures were also observed containing up to 8000cells, which were more easily spread by tissue maceration (FIG. 5B). Inaddition, many diverse cells and groups were interspersed among thecrypts and crypt-like structures (FIG. 5C). Some structures appeared tobe growing toward full-sized normal crypts containing ˜250, ˜500 or 1000cells (The spreading technique employed generally permitted exact cellcounts in structures of up to several hundred cells). Many cell groupswere seen as “rings” of exactly 8, 16, 32, 64 and 128 cells each. (FIG.5D). Higher magnification examination revealed that while most of thecells of the walls of the crypt-like structures had spherical or ovalnuclei as in the normal adult colonic crypt, colonies of cells witheither oval, cigar-shaped or bullet-shaped nuclei appeared at crypt wallbreaches. Colonies with oval and cigar-shaped nuclei had been observedin fetal gut but the “bullet-shaped” nuclear morphotype was seen only inadenomas and adenocarcinomas (FIG. 5E).

The “bullet-shaped” nuclear morphotype also appeared to arise frombell-shaped nuclei by asymmetrical amitoses with the irregular end(“bitten off”) emerging first. Small colonies of cells withbullet-shaped nuclei were seen and these colonies contained cellsundergoing ordinary mitoses save for the interesting fact that thecurious nuclear morphology was retained from early prophase throughanatelophase.

While rare in the normal adult colon the bell-shaped nuclei wereobviously playing an important role in adenomas. They appeared in anumber of adenoma contexts. Some were found as one to ten or more“bells” in the spaces among the crypt-like structures as shown in FIG.5D. Others were found as single “bells” in multicellular ring structuresin which one bell nucleus was always seen in the ring with 2n−1 cells ofspherical or other morphology as in FIGS. 5C and 5D. In the smaller tofull-sized structures that cohered under spreading conditions as didnormal colonic crypts, bell-shaped nuclei appeared as single bells, moreoften as a pair of bells or occasionally 4 or 8 bells within thecrypt-like structures basal cup. In the much larger irregular crypt-likestructures, bell-shaped nuclei were anatomically integrated into thewalls of the aberrant structures mixed with cells of other nuclearmorphologies. It appeared as if these larger irregular crypt-likestructures were mosaics of multiple different kinds of clusters eachwith it's own nuclear morphotype.

The bell-shaped nuclei of the adenomas differed in another remarkableway from the other nuclear morphotypes and from bell-shaped nuclei inembryos: though just more than a thousand bell-shaped nuclei have beenobserved in individual adenomas not a single bell-shaped nucleus in anyadenoma has been observed in the symmetrical amitotic form found infetal sections. It is also notable that no bell-shaped nucleus in acondition that could be described as pyknotic was observed among theadenomas scanned. Nuclei of all other morphological forms werefrequently found in mitosis or pyknosis at roughly equal frequencies(about 1-5/1000 total nuclei) when whole sections were scanned. Smallsubsections varied greatly with regard to mitotic and pyknotic counts.As used herein, “pyknotic” refers to a condition of nuclear rupture andchromatin condensation in irregular clumps that is indicative of a dyingcell.

Adenocarcinomas

While adenocarcinomas had much greater masses and many individualsections resisted spreading by gentle pressure, the admixture of crypts,larger irregular crypt-like structures and inter-crypt presence of ringsof 16, 32, 64 and 128 cells was essentially the same as in the muchsmaller adenomas. Breaches in the walls of the crypt-like structureswere associated with colonies of cells with identical nuclearmorphologies persisting into the three dimensional distributions ofcondensed prophase chromosomes. Bell-shaped nuclei were still found assinglets, pairs or larger numbers in the basal cup of crypts andembedded in complex whorls in the walls of the larger aberrantcrypt-like structures. FIGS. 6A-6C show a series of these bell-shapednuclei all typical of what is seen in adenocarcinomas. The set ofnuclear morphotypes in the adenocarcinomas appear to be identical withthe set seen in adenomas. In particular the “bullet-shaped” nuclearmorphotype not observed in fetal sections or adult colons was frequentlyobserved.

A discernible difference between adenomas and adenocarcinomas was thatthe orientation of the crypt-like structures was haphazard with base tolumen directions apparently randomly oriented with regard to the tumorsurface. Also crypts and irregular crypt-like structures were not foundfrequently in the tumor interior, which may be better characterized asan eclectic but not chaotic collection of smaller, locally organizedstructures. It is possible that these interspersed colonies of cellswith different nuclear forms represent a symbiotic community.

An important characteristic by which the adenocarcinomas differed fromadenomas was the frequent appearance of apparently organized groupingsof hundreds of bell-shaped nuclei, many of which were frequently (˜1%)involved in symmetrical amitoses. At low magnification these appeared inthe spaces among crypt-like structures and looked like a spider web orleaf vein skeleton. At higher magnification the thin “veins” were foundto be partially ordered strands of cells with bell-shaped nuclei havingthe curious characteristic of having their “mouths” oriented in the samedirection, 90° from the axis: the shoulder-to-shoulder orientation (FIG.6C). Furthermore, scanning through multiple sections of adenocarcinomasuncovered bell-shaped nuclei in the “head-to-toe” orientation observedin the fetal gut but not in the adenomas. These linearly arrayed nucleiwere also encased in the tubular structure (syncytium) seen in thefetus. An occasional pyknotic figure that might have been a bell-shapednucleus was observed but at a much lower frequency than symmetricalamitoses among bell-shaped nuclei.

Further Observations

Metastases of colorectal tumors in the liver recreate the pattern ofnuclear morphotypes, crypts and crypt-like structures seeminglyindistinguishable from adenocarcinomas. Scanning sections of sections ofadult human liver has revealed occasional bell-shaped nuclei as wereobserved in adult colonic crypts. Scans of adult mouse colons similarlyrevealed cells with bell-shaped nuclei in crypt bases as in adulthumans. In a potentially important observation for growth and study ofcultured cells with bell-shaped nuclei, a low frequency, ˜1/10,000cells, have been found in mouse cell cultures in which stem-likebehavior has been postulated and studied with regard to symmetrical andsymmetrical cell division kinetics (Sherley, J. et al., 1995, Proc.Natl. Acad. Sci USA, 92:136-140; Merok, J. et al., 2002, Cancer Res.,62:6791-6795).

Example 3

Post-embryonic organizing stem cells of the fetal colon werespecifically identified by an opened-mouth, bell-shaped nuclearmorphotype. These peculiar nuclei undergo both symmetric and asymmetricnuclear fission without general chromosome condensation. Nuclearfissions drive net growth and differentiation throughout fetal, neonataland juvenile life before a final metamorphosis into adult maintenancestem cells. These bell-shaped nuclear morphotypes are rarely found inadult colonic crypt bases but reappear prominently in preneoplasia andneoplasia and appear to drive net growth and differentiation in colonadenomas, adenocarcinomas and metastases.

Combining these observations with inferences derived from analyses ofhistorical age-specific colorectal cancer rates, present dayage-specific colonic adenoma prevalence and direct measurements ofgenetic change in human tissues suggests a default hypothesis forlate-onset carcinogenesis in the colon and perhaps other sites:oncomutations required for tumor initiation occur at markedly higherrates in juvenile stem cells than in adult maintenance stem cellsbecause of their peculiar DNA biochemistry and mode of segregation. Thehigher juvenile mutation rates have the effect of limiting tumorinitiation events to the juvenile years. Initiated juvenile stem cellscontinue to create local patches of juvenile tissue eventually observedin the colon as polyps and these cells maintain the “mutator” phenotypeimputed to juvenile cells. Additional oncomutation(s) and/or localbiochemical conditions during the slow but inexorable growth of thepreneoplastic colony switch one of the preneoplastic “juvenile” stemcells to a fetal stem cell phenotype that rapidly creates a lethal tumormass.

Organogenesis

The idea that organogenesis is accomplished by a linear cascade of stemcells capable of net growth by self propagation in symmetrical divisionsand responsible for differentiation by asymmetric divisions giving riseto a stem cell and an alternate cellular form is generally held bydevelopmental biologists. Confusion arises when attempts are made todifferentiate the identities and functions of the various forms andpotentials of cells from the early embryonic stem cells of theblastocyst capable of giving rise to viable embryos on transplantationand stem cells such as those isolated from the bone marrow of animalscapable of repopulating an hematopoietic system and possibly otherorgans. Adult stem cells or maintenance stem cells are posited to beresponsible for repopulation of the many tissue elements that turn overin organs such as the colon. In adults one may imagine smalldepositories of stem cells capable of repopulating tissues and organs ondemand. One can also imagine a different form of adult maintenance stemcell that defines clonal turnover units by intermittent asymmetricaldivisions that produce an initial transition cell that is lost bysubsequent binary divisions to secondary transition cells up todifferentiated terminal cells.

Disclosed herein are structures containing easily identifiable nuclearforms that appear to undergo both symmetric and asymmetric nuclearfission. These forms are identified by their bell-shaped nuclearmorphotypes that comprise some 30% of all nuclei of human gut in 5-7week fetuses but are found in the basal apex in somewhat less than 1% ofadult colonic crypts.

Carcinogenesis

The idea that carcinogenesis is accomplished by a linear cascade of stemcells capable of net growth by self propagation in symmetrical divisionsand responsible for differentiation by asymmetric divisions to createthe heterogeneity evidenced in adenocarcinomas is less widely held. Theconcept of carcinogenesis as “loss of cellular control” accompanied byseemingly random expression of genes such as those expressed in earlyembryogenesis is fairly widespread. However, dilution andtransplantation experiments, similar to those demonstrating the physicalexistence of organ restoring stem cells in hemoleukopoiesis haveestablished the existence of a very small fraction of tumor cells havingthe ability to give rise to a growing tumor containing a variety of celltypes. These practical demonstrations of the existence of tumor stemcells requires reflection on the idea of cancer as a highly degeneratestate and probing the possibility that it, like organogenesis, is theexpression of specific changes that define a pathway from normal stemcells to tumor stem cells. Relevant to this line of thought is thefinding of bell-shaped nuclei identical to those found in colonicembryogenesis in both colonic polyps (adenomas, preneoplastic lesions)and tumors (adenocarcinomas, neoplasia) and subsequent metastases.Preliminary enumeration of these nuclear forms and their frequency ofsymmetrical nuclear divisions permit comparison to the expected lowdivision rates of preneoplasia and rapid division of neoplasia.

Relationship of stem cell biology to age-specific cancer rates.

It is reasonable to infer the hypothesis that initiated stem cells growat near juvenile rates to form a preneoplastic colony (adenoma, polyp)from which inexorably emerged a neoplastic stem cell that grows at nearfetal rates to form a lethal tumor. The hypothesis that neoplasia is are-expression of the fetal phenotype is not original with us beingascribed to mid 19^(th) century pathologists such as Cohnheim (1875,Virchows Arch., 65:64; 1877-1880, Vorelesungen uber allgemeinePathologie. Ein Handbuch fur Artzte and Studierende. Berlin, Hirchswald1-2 691 S). The hypothesis arises that preneoplasia, adenomas or polypsin the human colon is a simple continuation of the phenotype of thejuvenile colon.

Data and derived inferences about the growth rate of preneoplasia wereaugmented by a fortuitous discovery about the growth rates of children'sweights as a function of age: male and female juveniles increase inaverage mass exponentially with a doubling time of about 6 years fromabout 1.5 years to 14.5 years in females and to 16.5 years in males. Thegrowth rate during these age intervals is 0.158 for males, 0.167 forfemales. Relating the growth rate of the colon to that of body massrequired modeling of the surface area of a cylinder inside a growingsphere that would increase as the mass^(2/3) or in this case,˜0.16^(2/3)˜0.11, a value equal to that estimated for the colonicpreneoplastic growth rate.

As the estimated growth rates of preneoplastic lesions of the colon areabout equal to the estimated growth rate of the juvenile colon, thehypothesis developed that preneoplasia in some way recreated conditionsof juvenile growth (Herrero-Jimenez et al., 2000). As net growth ofjuvenile stem cells would presumably be required for juvenile tissuegrowth, the idea arose that adenomas, which contain many of thehistological attributes of organized colon, might in fact be equivalentto patches of juvenile tissue expansion in a background of non-growingadult crypts. This hypothesis has been tested by computations limitingthe age of initiation to the age of maximum body mass and found that itis indeed possible to derive sets of parameters that accord with theage-specific colorectal cancer rates if the process of initiation islimited to the juvenile period. Indeed parameters can be derived thatfit the cancer rate data if initiation is limited to age five or lower.Such calculations demonstrate that the hypothesis of limitation ofinitiation to within the juvenile period is consistent with theage-specific cancer rate data but do not, however, demonstrate thehypothesis' validity.

Age-Specific Detection of Adenomas.

These theoretical constructs would remain in the domain of untestablehypotheses were it not for an important set of clinical observationsthat seem to bear directly on the hypothesis that tumor initiation isbounded by the juvenile period. In studies designed to define theoptimal age and number of proctoscopic examinations to detect and removepotentially neoplastic colonic adenomas, the fraction of persons withadenomas detectable by flexible sigmoidoscopy was found to reach astable maximum at about sixty years of age. When observations byproctologists with consistently high records of adenomas detection wereanalyzed, ˜15% of males and some ˜10% of females had somewhat more thanone adenoma on average. From these clinical data, it is reasonable toinfer: first, the slowly growing preneoplastic adenomas must have hadtheir origins far earlier in life than age sixty (with a growth rate of0.11 a tissue stem cell initiated at age 1 would have increased to only2⁷=128 preneoplastic stem cells by age 63. The number of total cells insuch an adenoma would be much larger on the order of a million totalcells); second, the similarity between the fraction of males withpolyps, ˜15%, and the estimated minimum fraction of males (1890s cohort)at lifetime risk of colon cancer, 18-20%, suggests that the individualswith adenomas at age sixty and those at lifetime risk may be identical(if this inference is confirmed on further analysis it would beimportant for two reasons: (a) it would eliminate any role for inheritedor genetic risk factors in the eventual transformation of preneoplasticlesions into neoplastic lesions in the colon, and (b) it would eliminateany important role for competing forms of mortality with risk factorsshared with colorectal cancer); third, the appearance of an average ofmore than one polyp in persons with polyps implies the male populationconsists of a subpopulation of some 20% in whom tumor initiation andpreneoplastic growth occur and a separate distinct subpopulation of some80% in which either tumor initiation and/or preneoplastic growth do notoccur; fourth, insofar as calculations of preneoplastic growth rates inmales and females yield identical results, the male/female ratio ofpersons with polyps over age sixty with polyps and the genderdifferences in the average number of polyps/individual with polypspermits the hypothesis that gender differences in cancer rates may beascribed entirely to the number of initiated colonic stem cells createdthrough the initiation susceptible juvenile or pre juvenile years (forinstance, if oncomutation rates were the same in males and females, theratio of expected preneoplastic colonies created in the juvenile yearswould simply be the ratio of stem cell years experienced up tomaturation at about 14.5 years in females and 16.5 years in males).

Histopathology.

Throughout the 20^(th) century, molecular, biochemical andhistolopathological analytical methods have been applied in parallel butindependent studies of tumors and embryos. Rich and extensive dataobtained showed that many normal ontogenic characteristics associatedwith the growth and development of humans re-appear much later in lifeas abnormal pathological growth that is cancers. At the beginning of21^(st) century scientists put forward the hypothesis that similaritybetween embryos and tumors (both monoclonal in origin, both appearing inexplicit heterogeneity of cells populations) can be explained from asingle point, that is the very specific cell such as a stem cell, cangive rise to a whole embryo, organs and tissues of an embryo as well asof a large tumor.

Table 1 shows the stem cells hierarchy in early human development.Between gestational age of 2 weeks (the stage of blastocyst andgastrulation) and 5-12 weeks (the stage of organogenesis) there is acascade of stem cells arising to correspond to each particular stage ofearly fetal development.

TABLE 1

Table 1. The hierarchy of stem cells arising through ontogenesis andthrough ‘blocking’ and-‘reversing’ mutagenesis of a stem cell to becomea cancer stem cell.

The importance of distinguishing among ontogenetically different stemcells has come from the disclosed cytogenetic evaluations of human fetaland adult tissues.

Embryonic stem cells of epiblast are the cells that are not committed toanything in pre-implanted embryo, divide by mitosis and their nuclei aresimply spherical in shape. They have no evidence of retainable polarityof the cell, nor of the nucleus. At the stage of elaboration of a bodyplan and organogenesis the cells acquire the polarity thought to benecessary to start spatially ‘directed’ migration of cells and celllayers to create bilaterally symmetrical body of a human, to outlinepositions of internal organs in a body cavity.

The middle stage of organogenesis of a gut (5-7 weeks), from which allmajor parts of a digestive system will arise, includes cells comprisingnuclei that are not just spherical but have diverse nuclear morphotypes.Nuclei of some of the cells have distinct polarity and divide not bymitosis but by the process more associated with “cup-from-cup” nuclearfission (FIG. 8). This particular observation has led to the conclusionthat the stem cells of organogenesis could be quite different from thatobserved in blastomeres of an embryo.

In postnatal growth of a child and later of a juvenile the epithelialtissue continue to grow by multiplication of the stem cell to increasethe number of turnover units per organ, and the number of cells perturnover unit until both features reach an organ-specific sizemaintained throughout life of an adult. For this matter, the stem cellstaking over the growth and function of organs in juveniles should bedistinguished from the stem cells of fetal growth and development asjuvenile stem cells. Embryonic, fetal and adult stem cells are differentin their capacity: embryonic stem cells are capable of creating thewhole organ or tissue by combining different cell lineages(pluripotency), while the adult stem cells can only give rise to a‘tissue-committed’ specific cells of a turnover units (multipotency).

If Sell's hypothesis of a ‘relationship between the stage ofdifferentiation of cancer stem cells and type of tumors’ is correct, theability to identify a single stem cell in it's ‘adult’, ‘juvenile’,‘fetal’ or ‘embryonic’ form would be a significant breakthrough in theanalysis of quantitative and qualitative characteristics of cancers(Sell, S., 2004. Crit. Rev. Oncol. Hematol., 51:1-28). Technical toolsto identify and collect a pure population of same type stem cells in atest tube are clearly useful.

Nuclei in the cells of developing fetal gut can be organized as a hollowbells (FIG. 8: a,b). Not only the cells with such nuclei are present at˜30% in the fetal gut but they have a very peculiar arrangement patternin the gut tissue: nuclei are oriented in one direction as “bell uponthe bell”. They are also enclosed in the structures resembling tubularsyncytia. In the normal crypts of adult colonic epithelium the cellswith bell-shaped nuclei are rare and, if they are present, their numberis not more than one per crypt. The cells with the same bell-shapednuclei appear in adenomas in larger numbers, in every crypt, normal oraberrant one (see FIG. 8: g,h) and then re-appear at high frequency inaberrant crypts and tumor masses of adenocarcinomas (see Table 1).

Cells with bell-shaped nuclei are observed in large numbers in fetuses,are rare in normal adult colon free of neoplasia, present, in smallnumbers (<1000) in adenomas of a few cubic millimeters and large numbers(>1,000,000) in adenocarcinomas of several cubic centimeters.

An argument that these cells could be stem cells have been supported byanother, also unusual, observations of bell-shaped nuclei divisions.Within the tubular syncytia the bell-shaped nuclei were giving rise toamazingly identical 3D copy of themselves, as if a Xerox™ copy of a‘template’ object. All stages of consequential separation of thebell-shaped nuclei (as a separation of a two paper cups), referred toherein as ‘nuclear fission’ because of the absence of nuclearcondensation to form mitotic chromosomes and typical mitotic apparatus,have been detected (FIG. 8: d). This finding has been followed by theobservations of all seven nuclear morphotypes previously found indeveloping human gut (FIG. 8: e,i) as emerging from the bell-shapednuclear, always in one direction: out of the ‘mouth’ of the bell. Cellswith different nuclear morphotypes are phenotypically different and onecan expect to detect the difference in gene expression and proteinsynthesis profiles between the cells with different nuclear morphotype.

The cells of an embryonic blastomere do not contain nuclei shaped ashollow bells; they have spherical nuclei and they are “embryonic stemcells”. At the same time the hollow bell-shaped nuclei are alreadyobserved as early as ˜5 weeks of gestation. The next stage in human lifeafter fetal gut development where cells with bell-shaped nuclei reappearin large quantities is a pathological condition of adult colon: colonadenoma. An explicit diversity of nuclear morphotypes is also observablein colon tumors, very similar to one in fetal colon. The cells andnuclear morphotypes diversity is another reflection of cells‘heterogeneity’ in tumors. Tumors are also characterized byhistopathologists as containing immature cells. At different stages indeveloping of a tumor a single juvenile stem cell in pre-neoplasia and asingle fetal stem cell in neoplasia can contribute to heterogeneousphenotype of both but with different fractions of immature cells (Table2).

TABLE 2 Stem cells and their qualities in embryogenesis andcarcinogenesis as observed in histopathological specimens of a colon.Pre-neoplastic Stem cell In fetal Adult normal lesion qualities 5-7weeks gut tissue (adenoma) Adenocarcinoma Differentiation The stem cellsof The adult stem The juvenile The stem cell of status organogenesis.cells stem cell organogenesis Tissue, organ High fraction Low Low butnot as Relatively high specific number (up to 30% in 5 weeks (~4 10⁻⁵)low as in (mean value gut) normal ~0.2%) epithelium (~2-4 10⁻³) Netgrowth rate Expansion of different cells Maintenance of First clonalMultiple clones types in organogenesis specific cells in appearance ofof cells adult crypt the cells ‘of morphologically fetal gut’ typeresembling those of fetal gut. Multiplication Frequently observed Notobserved Not observed Observed but and self-renewal in fetal gut morerare then by asymmetrical symmetric types of divisions division Givingrise to a Frequently observed in fetal Not observed Infrequent Morefrequent a different cell gut asymmetrical asymmetrical lineagesdivisions divisions (asymmetric observed observed division) Cell and/orDistinct polarity of the The nuclei in Orientation of Probably definesnuclear polarity nuclear is observable crypts niches the nuclei can an‘inward’ have ‘single way’ differ invasive tumor's orientationthroughout growth crypts

Example 4

Described herein are methods to characterize nuclear structures, DNAcontent and the spatial distribution of chromosomes in bell-shapednuclei of cells and syncytia by quantitative image cytometry and/orconfocal microscopy. These methods allow for the discovery of DNAcontent and/or chromosome distribution vary among bell-shaped nuclei ofdiffering morphology, tumor type (colonic vs. pancreatic) and nicheswithin tumors. They also allow for the characterization of the progressof DNA synthesis and presence of proteins associated with mitosis inbell-shaped nuclei during symmetrical and the several forms ofasymmetrical nuclear fission.

Also described herein are methods useful in isolating cells and syncytiawith bell-shaped nuclei as homogeneous samples. Such methods include theuse of “catapult” pressure activated laser micro-dissection to createsamples of cells homogeneous for nuclear morphology that may be appliedto analyses of metabolites and macromolecules. The methods for isolatingsingle cells allow for the discovery of means to recognize nuclearmorphology in unfixed tumor preparations so that homogeneouspreparations of live cells and syncytia with bell-shaped nuclei can bestudied ex vivo. Live bell-shaped nuclei or cells containing them couldthen be studied to better understand their peculiar DNA synthetic andsegregation mechanisms and suggest means to interfere with theseprocesses in cancer therapies.

Described herein is an exploration of how bell-shaped nuclei arespatially organized, how chromatin is dispersed in the nuclei, whetheror not specific chromosomes occupy specific territories throughout theinterior of nuclear lamina might suggest more specific therapeutictargets research and provide additional understanding of therelationship between nuclear morphotype (shape) and gene expression.Such exploration can be performed by existing methods ofimmunocytochemistry, molecular biology and related sciences.

An array of distinct closed nuclear forms has been found in fetalhindgut, colonic adenomas and adenocarcinomas that appear to arise abinitio from asymmetrical nuclear fission from bell-shaped nuclei butsubsequently divide by mitosis and die by apoptosis. The shared set ofnuclear forms in fetuses and tumors that are absent in adult tissuesupport the 19^(th) century hypothesis that tumors were “embryonic”growths in adult organs (FIG. 9). These findings coupled with computerbased image analysis and laser-assisted micro-dissection make therecognition and collection of large numbers of cells with specificnuclear morphologies possible. The characterization of the arrays ofmolecular and biochemical constituents in cells with bell-shaped nucleias well as cells of other nuclear morphotypes peculiar to tumors shouldprovide a previously unexpected set of potential therapeutic targets.

Oncogenesis like ontogenesis appears to proceed by lineal descentthrough an expanding set of stem cells. Only a small fraction of cellsfrom a human tumor have the capacity to form new tumors as xenografts inimmuno-suppressed rodents. Limiting dilution xenograft experiments haveshown that the putative tumorigenic cells displayed stem cell-likeproperties in that they were capable of generating new tumors containingadditional stem cells as well as regenerating the phenotypically mixedpopulations of cells present in the original tumor (Singh, S. et al.,2004. Oncogene, 23:7267-7273; Clarke, M., 2005. Biol. Blood MarrowTransplant., 11:14-26).

Nearly all forms of late onset cancer pass through an extended period ofpreneoplasia and these preneoplastic colonies are monoclonal resultingfrom more than one rare genetic mutation from the germinal DNA. By thebeginning of 21st century, direct attempts to enrich tumor cellpopulations with stem cells for transplant/dilution experiments haddemonstrated that not only were tissue stem cells the likely cells oforigin of preneoplasia but tumors themselves contained stem cells(Pardal, R. et al., 2003. Nature Rev., 3:895-902). Modern restatement ofthe hypothesis that tumors are in fact reasonably well organizedheterogeneous fetal structures has been reviewed and extended (Sell, S.,2004. Crit. Rev. Oncol. Hematol., 51:1-28). ‘Carcinoembryonic’ stemcells would be expected to increase in number and give rise todifferentiated cell types populating the highly heterogeneous nicheswithin the tumor mass.

Various antigenic markers employed throughout the stem cell field havebeen used to enrich for cells capable of regenerating tissues or tumorsoften to a high degree, but no cells within these enriched populationshave demonstrated any microscopic morphological cellular property thatmarks them as stem cells. If it is true that tumors arise from a singlestem cell, a means is required to identify them and to collect them ashomogeneous population of stem cells sufficient for analysis ofmolecular and biochemical analytes.

One method to achieve this goal relies on laser capture microdissection(LCM) to select and collect the tens of thousands of cells necessary formacromolecular array analyses and homogeneous with regard tomicrohistological properties that identify tumor stem cells.Alternately, dispersed cells with stem cell-associated surface markersof tumors have been enriched by flow cytometry and cell sorting fromheterogeneous cell populations (Morrison, S. et al., 1999. Cell,96:737-749; Suzuki, A. et al., 2004. Diabetes, 53:2143-2152).

The primary targets of existing methods of cancer therapeutics are cellstransiting the cell cycle (Gomez-Vidal, J. et al., 2004. Curr. Top. Med.Chem., 4:175-202; Fischer, P. and Gianella-Borradori, A., 2005. ExpertOpin. Investig. Drugs, 14:457-477). No distinction is made between cellsin transit between adult maintenance stem cells that divide to providetransition cells to replace the loss of terminal cells lost byprogrammed cell death and tumor stem cells. Therapy aims at the narrowwindow of regimens that kill all tumor stem cells without killing thepatient, but adult maintenance stem cells would logically be expected tohave the property of zero net cell growth while tumor stem cells, likefetal stem cells, are by definition involved in rapid net cell growth.Adult maintenance stem cell divisions would seem per force to beasymmetrical in nature giving rise to a new maintenance stem cell and afirst differentiated transition cell. Tumor stem cells would requiresuccessive symmetrical nuclear divisions to support net tumor growth. Itis in the discovery of bell-shaped nuclei undergoing symmetrical‘cup-from-cup’ nuclear division in tumors that a specific target forcytostatic or cytocidal therapies has been identified.

One theory lending support to this is the theory that tumors could byasphyxiated by preventing angiogenesis (Folkman, J. and Ingber, D.,1992. Sem. Cancer Biol., 3:88-96). Creating hypoxia may recreate theconditions of early embryogenesis so far as tumor stem cells areconcerned and may explain the palliative but not curative effects ofanti-angiogenic tactics. Blocking differentiation in tumors may blockdifferentiation in normal tissues with undesirable consequences.Understanding that current cancer therapies are only minimally effectivebecause the stem cells can repopulate tumors in a short period of timehas become a powerful stimulus to the search for molecular andbiochemical characteristics peculiar to tumor stem cells as opposed toadult maintenance stem cells. Such molecular and/or biochemicalcharacteristics of tumor stem cells serve as targets in cancertherapeutics.

A key aspect of testing such theories and characterizing (molecular andbiochemical properties) neoplastic stem cells and other neoplastic celltypes is their isolation in sufficient numbers and degree of purity topermit quantitative chemical and biochemical analyses. Several means canbe employed to obtain such cellular isolates. Laser capturemicrodissection, for example, is a method known in the art that allowsone of skill in the art to manually select and microdissect batches of10,000 bell-shaped nuclei from undifferentiated niches of colonic andpancreatic tumors. Cells ‘catapulted’ into the receiving vial are spreadon microscope slides and scored as “bell”, “not-bell” or “indeterminate”on the basis of morphology. When a reasonable level of enrichment isreached (>75% of isolated nuclei scored as “bell”), the procedure isvaried to preserve mRNA, proteins etc. for further analyses.

Existing protocols to isolate live colonic epithelium cells isolationhave been adapted for continued observations in cell culture thatpreserve their structural and functional characteristics (Stich, M. etal., 2003. Pathol. Res. Pract., 199:405-409; Micke, P. et al., 2004. J.Pathol., 202:130-138). Isolated cells or syncytia with bell-shapednuclei can be placed in slowly-stirred microcarrier flasks with varyingoxygen concentrations to mimic the oxygen levels expected in earlyembryos and unvascularized tumor niches. Medium replete with glutaminebut lower in glucose than “standard” cell culture media formulations isalso used to encourage growth of cells and syncytia with thesebell-shaped nuclei so they can be studied under controlled laboratoryconditions. Application of these methodologies is reasonably expected toyield a homogeneous cellular preparation with regard to nuclearmorphology, mode of division and/or presence in special forms ofmulticellular aggregates or syncytia. These cells can also be isolatedin a manner such that they can be propagated and studied in cell ortissue culture.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for preparing a mammalian tissue sample suitable for the identification of cells comprising bell-shaped nuclei, comprising: a) disrupting cellular adhesions of cellular sheets of the tissue sample; b) spreading the cells with disrupted adhesions onto a hard surface; and c) artificially staining the cells and/or macromolecules of the sample, thereby allowing visualization of nuclei, wherein the structural integrity of the nuclei of the cells remains intact, thus rendering the sample suitable for the identification of bell-shaped nuclei.
 2. The method of claim 1, wherein the tissue sample forms a layer on the microscope slide of about 0.5 millimeters.
 3. The method of claim 1, wherein the tissue sample forms a layer greater than about 50 microns.
 4. The method of claim 1, wherein the sample is a tissue sample obtained by surgical excision.
 5. The method of claim 1, wherein the sample is physically or chemically fixed prior to cellular degradation of nuclei.
 6. The method of claim 5, wherein the sample is frozen.
 7. The method of claim 5, wherein the sample is treated with one or more chemical fixing agents selected from the group consisting of: alcohols, aldehydes, organic acids and combinations thereof.
 8. The method of claim 7, wherein the fixing agent comprises methanol and acetic acid.
 9. The method of claim 1, wherein the cells of the sample are partially dissociated by tissue maceration and spreading.
 10. The method of claim 1, wherein DNA is stained, thereby allowing visualization of nuclei.
 11. The method of claim 4, wherein the tissue sample is fixed within 30 minutes of surgical removal.
 12. The method of claim 1, wherein the tissue sample is obtained from a mammal.
 13. The method of claim 12, wherein the mammal is selected from the group consisting of: primates, rodents, canines, felines, porcines, ovines, bovines and rabbits.
 14. The method of claim 13, wherein the mammal is a human.
 15. The method of claim 1, wherein the class or classes of nuclear morphotypes further comprise cigar-shaped, condensed spherical, spherical, oval, sausage-shaped, kidney-shaped, and bullet-shaped nuclei.
 16. The method of claim 1, wherein the nuclei are contained in multinuclear syncytia or in mononuclear cells.
 17. The method of claim 1, wherein the presence of non-spherical and non-oval nuclei in blood vessel wall tissue is indicative of an incipient atherosclerotic condition. 